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Expression of senescence-related genes in human corneal endothelial cells.

Song Z, Wang Y, Xie L, Zang X, Yin H - Mol. Vis. (2008)

Bottom Line: The results of the young (< or =30 years) and the old (> or = 50 years) were compared by the unpaired t-test.There was no significant difference in the expression levels of p21(WAF1/CIP1), p27(KIP1), and p53 between the young and old donors (p=0.875, 0.472, and 0.430, respectively).The p16(INK4a) signal pathway might play a key role in the process of senescence in HCECs.

View Article: PubMed Central - PubMed

Affiliation: State Key Lab Cultivation Base, Shandong Provincial Key Lab of Ophthalmology, Shandong Eye Institute, Qingdao, China.

ABSTRACT

Purpose: To investigate the expression of p16(INK4a), p21(WAF1/CIP1), p27(KIP1), and p53 in human corneal endothelial cell (HCEC) senescence ex vivo at various donor ages.

Methods: Residual corneal tissues obtained after penetrating keratoplasty were used in this study. Age, death-to-preservation interval, and preservation-to-surgery interval of the donors were recorded. Corneal endothelial cell survival and density were evaluated by trypan blue and alizarin red staining immediately after keratoplasty. Fresh frozen sections of donor corneas at various ages (18, 33, 54, and 68 years) were immunostained in situ. Total RNA extracted from age groups of 20, 30, 40, 50, and 60 years was evaluated by reverse-transcriptase polymerase chain reaction (PCR) to reveal the expression of the senescence-related genes, p16(INK4a), p21(WAF1/CIP1), p27(KIP1), and p53, in HCECs. Total RNA extracted from 20-, 24-, 26-, 30-, 50-, 55-, 56-, and 60-year-old donor groups was subjected to real-time PCR analysis for measurement of gene expression. The results of the young (< or =30 years) and the old (> or = 50 years) were compared by the unpaired t-test. Ex vivo senescence of HCECs from the donors at various ages (9, 17, 23, 57, 65, and 67 years) was observed by senescence-associated beta-galactosidase activity (SA-beta-Gal) staining at pH 6.0.

Results: The mean endothelial cell density of the donor corneas was 2,391.4+/-84.6 cells/mm(2), and the survival rate of the endothelial cells was 84.4%+/-5.3%. Hematoxylin and eosin staining showed normal structures of the corneal epithelium, stroma, and endothelium. The expression and nuclear localization of p16(INK4a), p21(WAF1/CIP1), p27(KIP1), and p53 in HCECs were confirmed by immunohistochemistry in situ. Reverse transcriptase PCR examination showed positive target bands of each gene at each age group. An age-related increase in p16(INK4a) expression was observed by real-time PCR (p=0.014). There was no significant difference in the expression levels of p21(WAF1/CIP1), p27(KIP1), and p53 between the young and old donors (p=0.875, 0.472, and 0.430, respectively). Strong SA-beta-Gal activity was observed in the endothelial cells of the old donors while there was weak and little-to-no blue staining in the endothelia from the young.

Conclusions: The population of HCECs exhibiting senescence-like characteristics increases with age. p16(INK4a), p21(WAF1/CIP1), p27(KIP1), and p53 are expressed in HCECs despite donor ages. The p16(INK4a) signal pathway might play a key role in the process of senescence in HCECs.

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Quantitative real-time polymerase chain reaction analyses of p27KIP1 mRNA expression in human corneal endothelial cells at various ages. Total RNA was isolated from HCECs of each group, and the first-strand cDNA were aynthesized from equal amounts of total RNA. Quantitative real-time PCR was performed to analyze p27KIP1 mRNA expression. A shows the expression of p27KIP1 from the eight age groups normalized to GAPDH mRNA. B demonstrates fold change in p27KIP1 expression relative to the calibrator of the 20-year-old group. C is the comparison of the average level of p27KIP1 mRNA expressed in HCECs between the young (≤ 30 years) and old (≥ 50 years) donors. Data are presented as mean±standard deviation, and a p<0.05 is considered to be statistically significant. Results indicate no significant increase (p=0.472) in p27KIP1 mRNA expression in HCECs from the old donors.
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f5: Quantitative real-time polymerase chain reaction analyses of p27KIP1 mRNA expression in human corneal endothelial cells at various ages. Total RNA was isolated from HCECs of each group, and the first-strand cDNA were aynthesized from equal amounts of total RNA. Quantitative real-time PCR was performed to analyze p27KIP1 mRNA expression. A shows the expression of p27KIP1 from the eight age groups normalized to GAPDH mRNA. B demonstrates fold change in p27KIP1 expression relative to the calibrator of the 20-year-old group. C is the comparison of the average level of p27KIP1 mRNA expressed in HCECs between the young (≤ 30 years) and old (≥ 50 years) donors. Data are presented as mean±standard deviation, and a p<0.05 is considered to be statistically significant. Results indicate no significant increase (p=0.472) in p27KIP1 mRNA expression in HCECs from the old donors.

Mentions: Total RNA extracted from HCECs of the eight age groups was subjected to quantitative real-time PCR for p16INK4a, p21WAF1/CIP1, p27KIP1, and p53. Figure 3 shows the expression of p16INK4a normalized to GAPDH RNA and the fold change in gene expression related to the calibrator of the 20-year-old group. There was a statistically significant difference in p16INK4a expression between the young (≤ 30 years) and old groups (≥ 50 years; p=0.014). Relative gene expression quantitation and fold change from the eight groups with p21WAF1/CIP1, p27KIP1, and p53 were shown in Figure 4, 5, and 6, respectively. There was no significant difference in p21WAF1/CIP1, p27KIP1, and p53 expression between any two age groups (p=0.875, 0.472, 0.430, respectively). HCECs from the old donors expressed significantly higher levels of p16INK4a than cells from young donors. In contrast, there were no obvious changes in the expression of p21WAF1/CIP1, p27KIP1, and p53 with age.


Expression of senescence-related genes in human corneal endothelial cells.

Song Z, Wang Y, Xie L, Zang X, Yin H - Mol. Vis. (2008)

Quantitative real-time polymerase chain reaction analyses of p27KIP1 mRNA expression in human corneal endothelial cells at various ages. Total RNA was isolated from HCECs of each group, and the first-strand cDNA were aynthesized from equal amounts of total RNA. Quantitative real-time PCR was performed to analyze p27KIP1 mRNA expression. A shows the expression of p27KIP1 from the eight age groups normalized to GAPDH mRNA. B demonstrates fold change in p27KIP1 expression relative to the calibrator of the 20-year-old group. C is the comparison of the average level of p27KIP1 mRNA expressed in HCECs between the young (≤ 30 years) and old (≥ 50 years) donors. Data are presented as mean±standard deviation, and a p<0.05 is considered to be statistically significant. Results indicate no significant increase (p=0.472) in p27KIP1 mRNA expression in HCECs from the old donors.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2254959&req=5

f5: Quantitative real-time polymerase chain reaction analyses of p27KIP1 mRNA expression in human corneal endothelial cells at various ages. Total RNA was isolated from HCECs of each group, and the first-strand cDNA were aynthesized from equal amounts of total RNA. Quantitative real-time PCR was performed to analyze p27KIP1 mRNA expression. A shows the expression of p27KIP1 from the eight age groups normalized to GAPDH mRNA. B demonstrates fold change in p27KIP1 expression relative to the calibrator of the 20-year-old group. C is the comparison of the average level of p27KIP1 mRNA expressed in HCECs between the young (≤ 30 years) and old (≥ 50 years) donors. Data are presented as mean±standard deviation, and a p<0.05 is considered to be statistically significant. Results indicate no significant increase (p=0.472) in p27KIP1 mRNA expression in HCECs from the old donors.
Mentions: Total RNA extracted from HCECs of the eight age groups was subjected to quantitative real-time PCR for p16INK4a, p21WAF1/CIP1, p27KIP1, and p53. Figure 3 shows the expression of p16INK4a normalized to GAPDH RNA and the fold change in gene expression related to the calibrator of the 20-year-old group. There was a statistically significant difference in p16INK4a expression between the young (≤ 30 years) and old groups (≥ 50 years; p=0.014). Relative gene expression quantitation and fold change from the eight groups with p21WAF1/CIP1, p27KIP1, and p53 were shown in Figure 4, 5, and 6, respectively. There was no significant difference in p21WAF1/CIP1, p27KIP1, and p53 expression between any two age groups (p=0.875, 0.472, 0.430, respectively). HCECs from the old donors expressed significantly higher levels of p16INK4a than cells from young donors. In contrast, there were no obvious changes in the expression of p21WAF1/CIP1, p27KIP1, and p53 with age.

Bottom Line: The results of the young (< or =30 years) and the old (> or = 50 years) were compared by the unpaired t-test.There was no significant difference in the expression levels of p21(WAF1/CIP1), p27(KIP1), and p53 between the young and old donors (p=0.875, 0.472, and 0.430, respectively).The p16(INK4a) signal pathway might play a key role in the process of senescence in HCECs.

View Article: PubMed Central - PubMed

Affiliation: State Key Lab Cultivation Base, Shandong Provincial Key Lab of Ophthalmology, Shandong Eye Institute, Qingdao, China.

ABSTRACT

Purpose: To investigate the expression of p16(INK4a), p21(WAF1/CIP1), p27(KIP1), and p53 in human corneal endothelial cell (HCEC) senescence ex vivo at various donor ages.

Methods: Residual corneal tissues obtained after penetrating keratoplasty were used in this study. Age, death-to-preservation interval, and preservation-to-surgery interval of the donors were recorded. Corneal endothelial cell survival and density were evaluated by trypan blue and alizarin red staining immediately after keratoplasty. Fresh frozen sections of donor corneas at various ages (18, 33, 54, and 68 years) were immunostained in situ. Total RNA extracted from age groups of 20, 30, 40, 50, and 60 years was evaluated by reverse-transcriptase polymerase chain reaction (PCR) to reveal the expression of the senescence-related genes, p16(INK4a), p21(WAF1/CIP1), p27(KIP1), and p53, in HCECs. Total RNA extracted from 20-, 24-, 26-, 30-, 50-, 55-, 56-, and 60-year-old donor groups was subjected to real-time PCR analysis for measurement of gene expression. The results of the young (< or =30 years) and the old (> or = 50 years) were compared by the unpaired t-test. Ex vivo senescence of HCECs from the donors at various ages (9, 17, 23, 57, 65, and 67 years) was observed by senescence-associated beta-galactosidase activity (SA-beta-Gal) staining at pH 6.0.

Results: The mean endothelial cell density of the donor corneas was 2,391.4+/-84.6 cells/mm(2), and the survival rate of the endothelial cells was 84.4%+/-5.3%. Hematoxylin and eosin staining showed normal structures of the corneal epithelium, stroma, and endothelium. The expression and nuclear localization of p16(INK4a), p21(WAF1/CIP1), p27(KIP1), and p53 in HCECs were confirmed by immunohistochemistry in situ. Reverse transcriptase PCR examination showed positive target bands of each gene at each age group. An age-related increase in p16(INK4a) expression was observed by real-time PCR (p=0.014). There was no significant difference in the expression levels of p21(WAF1/CIP1), p27(KIP1), and p53 between the young and old donors (p=0.875, 0.472, and 0.430, respectively). Strong SA-beta-Gal activity was observed in the endothelial cells of the old donors while there was weak and little-to-no blue staining in the endothelia from the young.

Conclusions: The population of HCECs exhibiting senescence-like characteristics increases with age. p16(INK4a), p21(WAF1/CIP1), p27(KIP1), and p53 are expressed in HCECs despite donor ages. The p16(INK4a) signal pathway might play a key role in the process of senescence in HCECs.

Show MeSH
Related in: MedlinePlus